Identification of the binding region of the [2Fe-2S] ferredoxin in stearoyl-acyl carrier protein desaturase: insight into the catalytic complex and mechanism of action.

Stearoyl-acyl carrier protein desaturase (Delta9D) catalyzes the O(2) and 2e(-) dependent desaturation of stearoyl-acyl carrier protein (18:0-ACP) to yield oleoyl-ACP (18:1-ACP). The 2e(-) are provided by essential interactions with reduced plant-type [2Fe-2S] ferredoxin (Fd). We have investigated the protein-protein interface involved in the Fd-Delta9D complex by the use of chemical cross-linking, site-directed mutagenesis, steady-state kinetic approaches, and molecular docking studies. The treatment of the different proteins with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide revealed that carboxylate residues from Fd and lysine residues from Delta9D contribute to cross-linking. The single substitutions of K60A, K56A, and K230A on Delta9D decreased the k(cat)/K(M) for Fd by 4-, 22-, and 2400-fold, respectively, as compared to wt Delta9D and a K41A substitution. The double substitution K56A/K60A decreased the k(cat)/K(M) for Fd by 250-fold, whereas the triple mutation K56A/K60A/K230A decreased the k(cat)/K(M) for Fd by at least 700 000-fold. These results strongly implicate the triad of K56, K60, and K230 of Delta9D in the formation of a catalytic complex with Fd. Molecular docking studies indicate that electrostatic interactions between K56 and K60 and the carboxylate groups on Fd may situate the [2Fe-2S] cluster of Fd closer to W62, a surface residue that is structurally conserved in both ribonucleotide reductase and mycobacterial putative acyl-ACP desaturase DesA2. Owing to the considerably larger effects on catalysis, K230 appears to have other contributions to catalysis arising from its positioning in helix 7 and its close spatial location to the diiron center ligands E229 and H232. These results are considered in the light of the presently available models for Fd-mediated electron transfer in Delta9D and other protein-protein complexes.

[1]  B. Fox,et al.  Methane monooxygenase from Methylosinus trichosporium OB3b. Purification and properties of a three-component system with high specific activity from a type II methanotroph. , 1989, The Journal of biological chemistry.

[2]  J. Lipscomb,et al.  Methane monooxygenase component B and reductase alter the regioselectivity of the hydroxylase component-catalyzed reactions. A novel role for protein-protein interactions in an oxygenase mechanism. , 1992, The Journal of biological chemistry.

[3]  H. Eklund,et al.  Di-iron-carboxylate proteins. , 1995, Current opinion in structural biology.

[4]  B. Fox,et al.  Peroxodiferric intermediate of stearoyl-acyl carrier protein delta 9 desaturase: oxidase reactivity during single turnover and implications for the mechanism of desaturation. , 1998, Biochemistry.

[5]  B. Fox,et al.  Lactose fed-batch overexpression of recombinant metalloproteins in Escherichia coli BL21 (DE3): process control yielding high levels of metal-incorporated, soluble protein. , 1995, Protein expression and purification.

[6]  J. Baldwin,et al.  Nature of the peroxo intermediate of the W48F/D84E ribonucleotide reductase variant: implications for O2 activation by binuclear non-heme iron enzymes. , 2004, Journal of the American Chemical Society.

[7]  John Shanklin,et al.  DESATURATION AND RELATED MODIFICATIONS OF FATTY ACIDS1. , 1998, Annual review of plant physiology and plant molecular biology.

[8]  I. Rayment,et al.  X‐ray structure of putative acyl‐ACP desaturase DesA2 from Mycobacterium tuberculosis H37Rv , 2005, Protein science : a publication of the Protein Society.

[9]  John D. Lipscomb,et al.  Dioxygen Activation by Enzymes Containing Binuclear Non-Heme Iron Clusters. , 1996, Chemical reviews.

[10]  Christopher C. Moser,et al.  Natural engineering principles of electron tunnelling in biological oxidation–reduction , 1999, Nature.

[11]  Gunter Schneider,et al.  Crystal structure of delta9 stearoyl‐acyl carrier protein desaturase from castor seed and its relationship to other di‐iron proteins. , 1996, The EMBO journal.

[12]  W. Koppenol,et al.  Binding of ferredoxin to ferredoxin: NADP+ oxidoreductase: The role of carboxyl groups, electrostatic surface potential, and molecular dipole moment , 1993, Protein science : a publication of the Protein Society.

[13]  S. Lippard,et al.  Structural Features of Covalently Cross-linked Hydroxylase and Reductase Proteins of Soluble Methane Monooxygenase as Revealed by Mass Spectrometric Analysis* , 2003, Journal of Biological Chemistry.

[14]  C. Lelong,et al.  Identification of the amino acids involved in the functional interaction between photosystem I and ferredoxin from Synechocystis sp. PCC 6803 by chemical cross-linking. , 1994, The Journal of biological chemistry.

[15]  J. Meyer,et al.  Specific interaction of the [2Fe-2S] ferredoxin from Clostridium pasteurianum with the nitrogenase MoFe protein. , 1997, Biochemistry.

[16]  G. Tollin,et al.  Structure-function relationships in Anabaena ferredoxin: correlations between X-ray crystal structures, reduction potentials, and rate constants of electron transfer to ferredoxin:NADP+ reductase for site-specific ferredoxin mutants. , 1997, Biochemistry.

[17]  B. Fox,et al.  Stearoyl-acyl carrier protein delta 9 desaturase from Ricinus communis is a diiron-oxo protein. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[18]  T A McKeon,et al.  Purification and characterization of the stearoyl-acyl carrier protein desaturase and the acyl-acyl carrier protein thioesterase from maturing seeds of safflower. , 1982, The Journal of biological chemistry.

[19]  Sandor Vajda,et al.  ClusPro: a fully automated algorithm for protein-protein docking , 2004, Nucleic Acids Res..

[20]  B. Oh,et al.  Protein expression, selective isotopic labeling, and analysis of hyperfine-shifted NMR signals of Anabaena 7120 vegetative [2Fe-2S]ferredoxin. , 1995, Archives of biochemistry and biophysics.

[21]  Pär Nordlund,et al.  The structure of Desulfovibrio vulgaris rubrerythrin reveals a unique combination of rubredoxin-like FeS4 and ferritin-like diiron domains , 1996, Nature Structural Biology.

[22]  Stephen J. Lippard,et al.  Crystal structure of a bacterial non-haem iron hydroxylase that catalyses the biological oxidation of methane , 1993, Nature.

[23]  J. Lipscomb,et al.  Transient intermediates of the methane monooxygenase catalytic cycle. , 1993, The Journal of biological chemistry.

[24]  H. Eklund,et al.  Structure and function of the Escherichia coli ribonucleotide reductase protein R2. , 1993, Journal of molecular biology.

[25]  G. Kachalova,et al.  A redox‐dependent interaction between two electron‐transfer partners involved in photosynthesis , 2000, EMBO reports.

[26]  B. Fox,et al.  Role of hydrophobic partitioning in substrate selectivity and turnover of the ricinus communis stearoyl acyl carrier protein delta(9) desaturase. , 1999, Biochemistry.

[27]  S. Lippard,et al.  Product bound structures of the soluble methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath): protein motion in the alpha-subunit. , 2005, Journal of the American Chemical Society.

[28]  J. G. Leahy,et al.  Evolution of the soluble diiron monooxygenases. , 2003, FEMS microbiology reviews.

[29]  L. Que,et al.  Mössbauer and EPR studies of the binuclear iron center in ribonucleotide reductase from Escherichia coli. A new iron-to-protein stoichiometry. , 1989, The Journal of biological chemistry.

[30]  Maarten Merkx,et al.  Dioxygen Activation and Methane Hydroxylation by Soluble Methane Monooxygenase: A Tale of Two Irons and Three Proteins. , 2001, Angewandte Chemie.

[31]  G. Tollin,et al.  Structure-function relationships in Anabaena ferredoxin/ferredoxin:NADP(+) reductase electron transfer: insights from site-directed mutagenesis, transient absorption spectroscopy and X-ray crystallography. , 2002, Biochimica et biophysica acta.

[32]  I. Vijay,et al.  A method for the high efficiency of water-soluble carbodiimide-mediated amidation. , 1994, Analytical biochemistry.

[33]  G. Wagner,et al.  NMR structure of the [2Fe-2S] ferredoxin domain from soluble methane monooxygenase reductase and interaction with its hydroxylase. , 2002, Biochemistry.

[34]  Sandor Vajda,et al.  ClusPro: an automated docking and discrimination method for the prediction of protein complexes , 2004, Bioinform..

[35]  B. Fox,et al.  Mössbauer studies of the formation and reactivity of a quasi-stable peroxo intermediate of stearoyl-acyl carrier protein Delta 9-desaturase. , 1999, Biochemistry.